Effect of serum depletion on centrosome overduplication and death of human pancreatic cancer cells after exposure to radiation.

The tumor microenvironment is one of the key factors affecting the cellular response to radiation; however, the influence of serum concentration on tumor radiosensitivity remains poorly understood. We recently discovered that gamma-irradiation of tumor cells causes centrosome overduplication, which may lead to lethal nuclear fragmentation through the establishment of multipolar mitotic spindles. In the present study, we investigated the effect of serum depletion on radiation-induced cell death in relation to the centrosome dynamics in human pancreatic cancer cells. Exposure of Capan-1 cells to gamma-irradiation resulted in a time-dependent increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death. Treatment of irradiated cells with serum depletion drastically accelerated centrosome overduplication and the formation of multipolar spindles, resulting in increased nuclear fragmentation and cell death. Cell cycle analysis of irradiated cultures revealed that the reduced serum level increased the population of cells arrested in the G2/M phase, which might be responsible for the abnormal centrosome accumulation. These findings suggest that serum concentration can influence radiation-induced cell killing through modulating cell cycle progression and possibly centrosome overduplication.

Correlation between centrosome abnormalities and chromosomal instability in human pancreatic cancer cells.

Chromosomal instability, characterized by abnormal numbers or structures of chromosomes, is a common feature of human cancers, but the mechanisms behind these changes are still unclear. Since centrosomes play a pivotal role in balanced chromosomal segregation during mitosis, we attempted to investigate the association between centrosome abnormalities and chromosomal instability in a large number of human pancreatic cancer cell lines. Immunofluorescence microscopy revealed centrosomes that were highly atypical with respect to their size, shape, and number in most cell lines. These abnormal centrosomes contributed to the assembly of multipolar spindles, resulting in defective mitosis and chromosome mis-segregation. Interestingly, a high frequency of centrosome defects inversely correlated with the growth rate of cells in culture. Fluorescence in situ hybridization revealed a dramatic variation of chromosome numbers in cell lines with the defective centrosome phenotype. Furthermore, a significant positive correlation existed between the level of centrosome defects and the level of chromosomal imbalances. These results indicate that centrosome abnormalities can lead to spindle disorganization and chromosome segregation errors, which may drive the accumulation of chromosomal alterations. Thus, defects in centrosome function may be an underlying cause of genetic instability in human pancreatic cancers.

Isolation and analysis of an aciclovir-resistant murine cytomegalovirus mutant.

An aciclovir (ACV)-resistant murine cytomegalovirus (MCMV) was isolated from the Smith strain and the mutant was analysed. Attempts were also made to identify directly the mutated gene. The 50% inhibitory concentration (IC(50)) of ACV for the mutant strain was approximately 30 times higher than that for the wild-type strain. The mutant strain was equally sensitive to ganciclovir (GCV), but slightly resistant to cidofovir (CDV) and foscarnet (PFA) when compared with the wild-type. Molecular analysis of the mutant strain revealed that a single base mutation of cytosine (C) to guanine (G) occurred at the 2476th nucleotide position in the DNA polymerase gene region, resulting in an amino acid substitution of proline (Pro) with alanine (Ala) at codon 826. The marker transfer experiment confirmed that this mutation conferred ACV resistance to MCMV. This mutation at codon 826 was easily identified by means of Hae III digestion of the selected PCR product and electrophoresis.

Down-regulation of p27Kip1 expression is required for development and function of T cells.

The proliferation of T cells is regulated in a development-dependent manner, but it has been unclear whether proliferation is essential for T cell differentiation. The cyclin-dependent kinase inhibitor p27(Kip1) is abundant throughout development in cells of the T cell lineage, with the exception of late stage CD4(-)CD8(-) thymocytes and activated mature T cells, both of which show a high rate of proliferation. The role of down-regulation of p27(Kip1) expression in T cell development and function has now been investigated by the generation and characterization of three strains of p27 transgenic mice that express the transgene at various levels specifically in the T cell lineage. The numbers of thymocytes at CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) stages of development as well as those of mature T cells in peripheral lymphoid tissues were reduced in transgenic mice in a manner dependent on the level of p27(Kip1) expression. The development of thymocytes in the transgenic strain in which p27(Kip1) is most abundant (p27-Tg(high) mice) appeared to be blocked at the CD4(-)CD8(-)CD25(+)CD44(low) stage. Peripheral T cells from p27-Tg(high) mice exhibited a reduced ability to proliferate in response to mitogenic stimulation compared with wild-type T cells. Moreover, Ag-induced formation of germinal centers and Ig production were defective in p27-Tg(high) mice. These results suggest that down-regulation of p27(Kip1) expression is required for the development, proliferation, and immunoresponsiveness of T cells.

A possible role for centrosome overduplication in radiation-induced cell death.

Radiotherapy plays a key role in the treatment of many tumors; however, the precise mechanisms responsible for radiation-induced cell death remain uncertain. We have reported previously that ionizing radiation induces centrosome overduplication in human tumor cells. The present study was designed to elucidate a possible link between centrosome dysregulation and radiation-induced cell death. Exposure to 10 Gy gamma-radiation resulted in a substantial increase in cells containing an abnormally high number of centrosomes in a variety of cell lines derived from different types of human solid tumors. These aberrant centrosomes contribute to the assembly of multipolar spindles, thereby causing an unbalanced division of chromosomes and mitotic cell death characterized by the appearance of multi- or micronucleated cells. An extensive analysis of a panel of 10 tumor cell lines revealed a positive correlation between the fraction of cells with multiple centrosomes and the fraction with these nuclear abnormalities after irradiation. When the centrosome overduplication was blocked by enforced expression of p21Waf1/Cip1, the radiation-induced lethality was drastically rescued. Taken together, these results indicate that centrosome overduplication may be a critical event leading to mitotic failure and subsequent cell death following exposure to ionizing radiation.

Aberrant cell cycle checkpoint function and early embryonic death in Chk1(-/-) mice.

The recent discovery of checkpoint kinases has suggested the conservation of checkpoint mechanisms between yeast and mammals. In yeast, the protein kinase Chk1 is thought to mediate signaling associated with the DNA damage checkpoint of the cell cycle. However, the function of Chk1 in mammals has remained unknown. Targeted disruption of Chk1 in mice showed that Chk1(-/-) embryos exhibit gross morphologic abnormalities in nuclei as early as the blastocyst stage. In culture, Chk1(-/-) blastocysts showed a severe defect in outgrowth of the inner cell mass and died of apoptosis. DNA replication block and DNA damage failed to arrest the cell cycle before initiation of mitosis in Chk1(-/-) embryos. These results may indicate that Chk1 is indispensable for cell proliferation and survival through maintaining the G(2) checkpoint in mammals.

Targeted disruption of Skp2 results in accumulation of cyclin E and p27(Kip1), polyploidy and centrosome overduplication.

The ubiquitin-proteasome pathway plays an important role in control of the abundance of cell cycle regulators. Mice lacking Skp2, an F-box protein and substrate recognition component of an Skp1-Cullin-F-box protein (SCF) ubiquitin ligase, were generated. Although Skp2(-/-) animals are viable, cells in the mutant mice contain markedly enlarged nuclei with polyploidy and multiple centrosomes, and show a reduced growth rate and increased apoptosis. Skp2(-/-) cells also exhibit increased accumulation of both cyclin E and p27(Kip1). The elimination of cyclin E during S and G(2) phases is impaired in Skp2(-/-) cells, resulting in loss of cyclin E periodicity. Biochemical studies showed that Skp2 interacts specifically with cyclin E and thereby promotes its ubiquitylation and degradation both in vivo and in vitro. These results suggest that specific degradation of cyclin E and p27(Kip1) is mediated by the SCF(Skp2) ubiquitin ligase complex, and that Skp2 may control chromosome replication and centrosome duplication by determining the abundance of cell cycle regulators.

Protective effects of hochu-ekki-to, a Chinese traditional herbal medicine against murine cytomegalovirus infection.

The innate immunity against murine cytomegalovirus (MCMV) at the early phase of infection is mediated by NK cells and macrophages. We studied the effects of hochu-ekki-to (HET), a traditional Chinese herbal medicine, on the regulation of innate immunity mediated by NK cells and macrophages. We found the oral administration of HET to increase both the number of leukocytes in the spleen and liver and the splenic NK cell cytotoxicity associated with the increased induction of serum IFN-alpha/beta after an MCMV infection but it had no effect on liver NK cells. However, no differences were found in the serum IL-12, IFN-gamma, TNF-alpha and nitric oxide (NO) production in the culture of macrophages between the HET- and PBS-treated mice on day 2 after MCMV infection. In addition, HET-treated splenic and peritoneal macrophages were found to show a higher intrinsic resistance against in vitro MCMV infection than that of PBS-treated mice. Therefore, the HET-induced effects on NK cells and macrophages selectively reduced the viral load in the spleen but not in the liver at an early phase of MCMV infection. HET may thus be useful in the treatment of human cytomegalovirus infection which commonly occurs in HIV-infected AIDS patients.

Adenoma arising from the cystic duct and extending to the confluence of the extrahepatic biliary tract.

We describe a rare case of adenoma with a few foci of severe atypia arising from the cystic duct in a 68-year-old woman, initially diagnosed by the presence of intracholecystic tumefactive sludge on abdominal ultrasonography. Endoscopic retrograde cholangiography (ERC) disclosed a tuberous filling defect at the orifice of the cystic duct. Pathology examination of the biopsied specimen obtained from ERC disclosed not a cancerous but an adenomatous lesion. Macroscopically, the resected specimen showed a superficially spreading and shallowly elevated lesion extending from the cystic duct to the common bile duct. Although a few sporadic foci of severe atypia were observed, microscopy did not reveal any cancer findings, but confirmed the tumorous lesion as benign adenoma, showing mild-to-moderate atypia. Postoperative immunohistochemistry revealed no expression of p53 protein. We briefly refer to the rarity of adenoma in the biliary tract and discuss the difficulty of differential diagnosis of neoplastic lesions in the extrahepatic biliary tract.

[An autopsy case of AIDS complicated with cytomegalovirus infection and multiple Kaposi's sarcoma].

Cutaneous Kaposi's sarcoma (KS) is a well-known complication of the acquired immunodeficiency syndrome. KS in the internal organs, however, is rare in Japan. We present here a 33-years-old Japanese homosexual man who had AIDS complicated with cytomegalovirus (CMV) infection and KS. He was found to be HIV-seropositive, when he was 31-years-old. He visited our hospital in June 1996 because of high fever. The peripheral blood CD4+ lymphocyte counts were 2 per cubic millimeter, and CMV antigenemia was noted (p65 antigen positive cells; 240/50,000 white blood cells). Thereafter he was successfully treated with parental ganciclovir. On admission, some brown-colored flat nodules were found on the skin, and the diagnosis of KS was made by skin biopsy. We administrated human chorionic gonadotropin (hCG) for the treatment of KS, but had no clinical response. In September 1996, he complained of severe cough, shortness of breath, and vomiting. A chest radiogram showed nodular lesions and pleural effusion in bilateral lungs. A computed tomography of his chest also revealed nodular and linear densities distributed along the bronchovascular bundles. The ultrasonic examination of his abdomen revealed a duodenal nodule. Both nodules in the lungs and duodenum were proved to be KS based on the autopsy findings. Intranuclear inclusionbodies pathognomonic for CMV infections were detected in the stomach and the colon.

Genetic analysis of a ganciclovir-resistant human cytomegalovirus mutant.

We isolated a ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) from a laboratory strain, AD169, and analysed the mutant. Attempts were also made to identify directly the mutated gene. The 50% inhibitory concentration (IC50) of GCV for the mutant strain was five times higher than that of the wild-type strain. The mutant strain showed similar sensitivity to phosphonoacetic acid and cidofovir as the wild-type strain. These data suggest mutation in the UL97 gene encoding for the phosphotransferase that phosphorylates GCV. Molecular analysis of the mutant strain revealed that a single base substitution of adenine by cytosine occurred at the 1796 nucleotide position of the UL97 gene region, resulting in the substitution of lysine by threonine at codon 599 in the UL97 gene product. Marker transfer experiment confirmed that this mutation conferred HCMV resistance to GCV. The mutation at codon 599 was easily identified by means of RsaI digestion of the selected PCR product.

Role of macrophages in acute murine cytomegalovirus infection.

It has been recognized that macrophages play an important role in controlling virus infection in experimental animal models. To evaluate the role of macrophages in acute murine cytomegalovirus infection, macrophages in the spleen and the liver were eliminated by an intravenous injection of liposomes containing a cytolytic agent, dichloromethylene diphosphonate. The depletion of macrophages led to a significant increase of virus titer in the spleen and lungs in both susceptible BALB/c and resistant C57BL/6 mice during the first three days after intravenous infection. In the spleen, the increase of virus titer in macrophage-depleted BALB/c mice was much greater than that in NK cell-depleted mice. These results suggest that macrophages contribute to protection mainly by the mechanisms which are independent of NK cells during the first three days after infection. The increase of virus titer in macrophage-depleted C57BL/6 mice was as great as that in NK cell-depleted mice because of the high contribution of NK cells to protection in C57BL/6 mice. In the liver in both strains of mice, the effects of macrophage depletion on virus titer were not as much as those in the spleen and lungs. Furthermore, the local depletion of peritoneal macrophages resulted in a great increase of virus titer in the spleen at three days after intraperitoneal infection. We conclude that macrophages greatly contribute to decreasing the virus load in some organs possibly through either or both intrinsic and extrinsic mechanisms in the early phase of primary infection with murine cytomegalovirus.

Early viral complications following CD34-selected autologous peripheral blood stem cell transplantation for non-Hodgkin's lymphoma.

A patient with non-Hodgkin's lymphoma who received a CD34-selected autologous peripheral blood stem cell transplant (PBSCT) developed cytomegalovirus retinitis, adenovirus-associated haemorrhagic cystitis (HC) and fatal herpes simplex virus pneumonia. Depletion of mature T cells from the graft and a persistent decrease in CD4+ lymphocytes following PBSCT may have predisposed this patient to such viral infections. Infusion of cryopreserved autologous PBSC (containing mature T cells) was effective for adenovirus-associated HC. Immunosuppression and resultant viral infections may affect patients receiving CD34-selected autologous transplantation.

[Laboratory diagnosis of cytomegalovirus, HHV-6 and HHV-7 infectious diseases].

Cytomegalovirus, human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) are members of subfamily Betaherpesvirinae of family Herpesviridae. After infection, these betaherpesviruses persistent for the rest of life. Those viruses are occasionally reactivated and shed either with or without clinical symptoms. The laboratory diagnosis of the viral infections is established by the following methods: (1) isolation of the virus from specimens; (2) detection of virus antigens, and/or viral nucleic acids; and (3) detection of seroconversion or a significant rise in antibody titer. Accurate and rapid laboratory diagnosis of the viral diseases is prerequisite to effective antiviral chemotherapy. Especially, it is most important to distinguish viral diseases to be treated from mere viral infections.

Comparison of cytomegalovirus (CMV) antigenemia and CMV in bronchoalveolar lavage fluid for diagnosis of CMV pulmonary infection after bone marrow transplantation.

A comparative cytomegalovirus (CMV) diagnostic study was carried out on 30 bone marrow transplant patients. Forty-three bronchoalveolar lavage fluid (BALF) samples from these patients were examined for CMV by viral culture, polymerase chain reaction (PCR), shell vial and cytology. In parallel, peripheral blood samples were subjected to CMV antigenemia assay. CMV was detected in 12 (27.9%) of the 43 BALF samples (10 samples in viral culture, 10 samples in PCR, eight samples in shell vial and three samples in cytology). The CMV antigenemia assay yielded a positive result for six samples. The rates of agreement between results of the CMV antigenemia assay and results of each of the BALF tests were as follows: 81.4% with viral culture, 76.7% with PCR, 86.0% with shell vial, and 88.4% with cytology. Although the sensitivity of the CMV antigenemia assay was inferior to the sensitive tests of BALF samples, statistically significant correlations were demonstrated between the CMV antigenemia assay, viral culture, shell vial and cytology. Although the CMV antigenemia assay was shown to be useful for detection of CMV, it may be necessary to confirm not only the sensitivity but also the specificity of this method for prevention of CMV disease after BMT.

Progressive retinitis-encephalitis due to ganciclovir-resistant cytomegalovirus associated with aplastic anemia.

A 29-year-old woman with aplastic anemia who complained of visual disturbances and pain in the right eyeball was diagnosed as having cytomegalovirus (CMV) retinitis based upon the characteristic retinal changes and isolation of CMV. She received treatment with ganciclovir (GCV), and the retinitis initially responded for several months. However, the patient was found to have CMV lesions in the left eye followed by neurological symptoms. The CMV isolated just before her death was approximately 20 times more resistant to GCV than that isolated previously, suggesting that the GCV-resistant CMV had developed during the long-term treatment with GCV.

Increased incidence of cytomegalovirus (CMV) infection and CMV-associated disease after allogeneic bone marrow transplantation from unrelated donors. The Fukuoka Bone Marrow Transplantation Group.

Cytomegalovirus (CMV) infection and CMV-associated disease were monitored using the CMV antigenemia assay in 72 patients who received allogeneic bone marrow transplantation (BMT), and their incidences were compared between related and unrelated donor transplant patients. The incidence of CMV infection after BMT was significantly higher in patients who received transplants from HLA-matched unrelated donors than from HLA-matched sibling donors (87% vs 53%, P < 0.05). CMV-associated disease developed in 73% of unrelated and in 14% of sibling donor transplant patients (P < 0.01). The peak levels of CMV antigenemia were significantly higher in unrelated donors than in sibling donor transplant patients (16 vs 1 CMV antigen-positive cells per 50000 WBCs, P < 0.01). The median number of CMV antigen-positive cells on first detection was also significantly higher in unrelated donor transplant patients (15 vs 1, P < 0.01). The detection of CMV antigen-positive cells preceded the development of CMV-associated disease in 18% of unrelated donor transplant patients, suggesting a lower predictive value of CMV antigenemia for subsequent CMV-associated disease in unrelated donor BMT. Careful monitoring and further studies are needed for the early diagnosis and prevention of CMV-associated disease in unrelated donor BMT.

[Foscarnet therapy for ganciclovir-refractory cytomegalovirus hepatitis in a patient who underwent bone marrow transplantation from an unrelated donor].

Cytomegalovirus (CMV) hepatitis refractory to ganciclovir treatment occurred after prolonged administration of ganciclovir in a 36-year-old woman with chronic myelogeneous leukemia who had undergone allogeneic bone marrow transplantation (BMT) from an HLA-identical unrelated donor. The number of CMV antigen-positive leukocytes in blood were well correlated with the serum levels of transaminases and the antigenemia assay was useful in monitoring CMV hepatitis. The patient was treated with foscarnet, a potent inhibitor of CMV DNA-polymerase, which led to rapid improvement of the CMV antigenemia and the transaminase concentrations. Foscarnet therapy should be considered for ganciclovir-resistant CMV disease in the setting of BMT.

Genetic analysis of a clinical isolate of human cytomegalovirus exhibiting resistance against both ganciclovir and cidofovir.

Both ganciclovir-sensitive and -resistant human cytomegaloviruses (HCMV) were isolated from a patient with aplastic anemia complicated with CMV retinitis and encephalitis. Ganciclovir-resistant clinical isolate, 93-1R, also showed cross-resistance against (s)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine (cidofovir). Molecular analysis of plaque-cloned strains revealed that a single nucleotide substitution at 2160 (C to T) resulted in amino acid substitution at codon 501 from leucine to phenylalanine in the DNA polymerase gene. This mutation at codon 501 was easily identified by means of AluI digestion of the selected PCR product. The same mutation existed in the DNA fragment amplified from the patient's brain, suggesting that cross-resistant mutant 93-1R caused encephalitis. Furthermore, ganciclovir-resistant 93-1R-3 replicated much faster and was released more efficiently into the culture medium than ganciclovir-sensitive 91-7S-1.